The Toxicologist - Society of Toxicology

675 Δ 9 -TETRAHYDROCANNABINOL DECREASES
ANTIGEN PRESENTATION ON DENDRITIC CELLS
AFTER INFLUENZA INFECTION IN VIVO AND TOLL-
LIKE RECEPTOR STIMULATION IN VITRO.
P. Karmaus 1, 3 , W. Chen 2, 3 , B. Kaplan 3, 4 and N. Kaminski 4, 3 . 1 Cell & Molecular
Biology Program, Michigan State University, East Lansing, MI, 2 Microbiology &
Molecular Genetics, Michigan State University, East Lansing, MI, 3 Center for
Integrative Toxicology, Michigan State University, East Lansing, MI and
4 Pharmacology & Toxicology, Michigan State University, East Lansing, MI.
Δ9-Tetrahydrocannabinol (Δ9-THC) alters the function of various cells of the immune
system, in part, by exhibiting activity at two identified cannabinoid receptors,
1 (CB1) and 2 (CB2). Dendritic cells (DC) link the innate to the adaptive immune
response and thus are viewed as the orchestrators of T cell responses. We
employed an in vivo influenza A/PR8/34 (PR8) model and an in vitro bone marrow
derived DC (bmDC) culture to assess the effects of Δ9-THC on DC function.
To determine the contribution of CB1 and/or CB2 to modulation by Δ9-THC, we
used mice devoid of CB1 and CB2 (CB1-/-CB2-/-). Three days post influenza
challenge, lung infiltrating DC populations were determined by FACS for maturation
(CD80 and CD86) and antigen presentation (MHC II). CD11b+CD11c+
conventional DC (cDC) were elevated in PR8 treated mice. Δ9-THC (75 mg/kg)
reduced CD86 and MHC II surface expression on cDC in lungs from WT but not
in CB1-/-CB2-/- mice. In vitro, CD11b+CD11c+ bmDC were generated and
treated with LPS (1 μg/mL) and ssRNA (25 μg/mL), which induced expression of
CD86 and MHC II. Compared to WT, bmDC from CB1-/-CB2-/- had greater
basal and induced levels of CD86 and MHC II, yet both surface markers were reduced
by Δ9-THC (10 μM) treatment in both genotypes. Δ9-THC affected neither
CD80 expression nor viability. In addition, we tested the ability of bmDC to
elicit T cell responses. While TLR agonists enhanced bmDC induced T cell proliferation
and IFN-γ secretion, concurrent Δ9-THC treatment of bmDC suppressed
both endpoints in a CB1/CB2-dependent manner. These studies suggest that DC
are targets for Δ9-THC-mediated suppression and that CB1 and/or CB2 are important
in regulating immune function and mediating Δ9-THC suppression of DC
function. (Support: NIHDA07908)
676 ANTI-INFLAMMATORY PROPERTIES OF
ENDOGENOUS CANNABINOIDS CAN BE
ATTRIBUTED TO INDUCTION OF A TRANSIENT
POPULATION OF IMMUNOSUPPRESSIVE MYELOID
DERIVED SUPPRESSOR CELLS THAT PROGRESS INTO
IMMATURE MACROPHAGES.
A. R. Jackson, V. Hegde, P. Nagarkatti and M. Nagarkatti. Pathology,
Microbiology, and Immunology, University of South Carolina, Columbia, SC.
Cannabinoids are a group of compounds that mediate their physiological and behavioral
effects by activating specific cannabinoid receptors. Cannabinoid receptor
1 (CB1) is primarily expressed in the CNS. In contrast, cannabinoid receptor 2
(CB2) is predominantly expressed on immune cells. In addition to the exogenous
cannabinoids found in the Cannabis plant, there are also endogenous cannabinoids
(endocannabinoids, ECs), such as 2-arachadonoyl glycerol(2-AG) and N-arachidonoyl-ethanolamine(anadamide,AEA).
The ECs also mediate their effects by activating
CB1 and CB2 receptors. Cannabinoids have been shown to act as potent immunosuppressive
agents and are used to mediate beneficial effects in a wide range of
inflammatory diseases. Previously we have shown that ECs induce Myeloid Derived
Suppressor Cells (MDSCs) that are CD11b+Gr-1+after a single i.p. administration.
In the current study, we further characterized these cells by functional and expression
profile. Functionally, these cells showed the ability to inhibit the T-cell proliferative
response. Upon further examination, the response was shown to be dependent
on the expression and function of Arginase-1. The mechanism of induction of
these cells was linked to CB1 signaling as shown by experiments with knockout
mice and pharmacological blocking. While the immunosuppressive MDSCs were
induced within 12 hours post-EC injection, it was noted that on day 4, there was
an increase in the proportion of cells that expressed Gr1-/Cd11b Lo/F480 Lo,
thereby suggesting their differentiation towards myeloid pathway. Together, these
experiments demonstrate that the anti-inflammatory properties of ECs can be explained,
at least in part, through induction of immunosuppressive MDSCs
(Supported in part by NIH grants R01ES09098, P01AT003961, R01ES019313).
146 SOT 2011 ANNUAL MEETING
677 Δ 9 -TETRAHYDROCANNABINOL MODULATES
ELICITATION AND FUNCTION OF CYTOTOXIC T
LYMPHOCYTE (CTL) DIRECTED AGAINST HIV GP120.
W. Chen 1, 4 , S. Pike 4 , P. Karmaus 2, 4 , B. Kaplan 4, 3 and N. Kaminski 3, 4 .
1 Microbiology & Molecular Genetics, Michigan State University, East Lansing, MI,
2 Cell & Molecular Biology, Michigan State University, East Lansing, MI,
3 Pharmacology & Toxicology, Michigan State University, East Lansing, MI and
4 Center for Integrative Toxicology, Michigan State University, East Lansing, MI.
Twenty-five percent of HIV patients use marijuana for appetite stimulation to attenuate
HIV-associated nausea and wasting syndrome. The effects of cannabinoids
on the immune systems of immunocompromised HIV patients are not well understood.
Δ 9 -Tetrahydrocannabinol (Δ 9 -THC) is the predominant psychoactive compound
in marijuana, and is known to modulate immune competence. Depending
on the magnitude of T cell activation, both positive and negative effects of Δ 9 -THC
on T cell functions, e.g. IL-2 production, have been observed. A surrogate in vitro
mouse model for presenting HIV gp120 antigens to T cells has been established to
study the effects of Δ 9 -THC on elicitation and function of antigen-specific CTL. A
gp120-expressing dendritic cell clone, DC2.4 gp120 C1, was generated to serve as
antigen presenting cells (APC) to elicit gp120-specific T cells. CD8 + T cell activation,
shown by up-regulation of an early T cell activation marker, CD69, at 6 hr
post the initiation of elicitation, and proliferation were observed. On day 5, CTL
were restimulated with gp120-expressing target cells, EL4 gp120 C135. CTL activity
was assessed by intracellular IFNγ staining. Higher percentages of IFNγ + CD8 + cells
were detected when EL4 gp120 C135 was used as target cells compared to parental
EL4, suggesting a gp120-specific response. Under current activation conditions, induced
IFNγ secretion was enhanced by Δ 9 -THC when added during the elicitation
phase. CD69 expression and proliferation of CD8 + T cells were also enhanced by
Δ 9 -THC, suggesting an effect on the early stage of T cell activation. Collectively,
these results indicate that DC2.4 gp120 C1 cells are capable of eliciting a gp120-specific
CTL response, and Δ 9 -THC can enhance the elicitation and function of
gp120-specific CTL. (Supported by NIH DA07908)
678 EFFECTS OF CANNABINOIDS ON ANTI-HIV-TAT
INDUCED ANTIBODY PRODUCTION.
E. Velez1, 3 , R. Dasgupta1 , N. E. Kaminski1, 2 and B. F. Kaplan1, 2 . 1Center for
Integrative Toxicology, Michigan State University, East Lansing, MI, 2Pharmacology and Toxicology, Michigan State University, East Lansing, MI and 3Chemistry, University of Puerto Rico at Cayey, Cayey.
Late stage Human Immunodeficiency Virus-1 (HIV) infection is characterized by
an immune response insufficiency, which manifests itself as susceptibility to infection
by the occurrence of, among others, Kaposi’s sarcoma or B-cell lymphoma.
The immune response elicited by several HIV proteins has been studied. Among
these is Tat, an HIV protein that facilitates and accelerates the transcription of most
of the HIV genome. Since many people infected with HIV use marijuana for appetite
stimulation, and marijuana compounds, such as Δ9-tetrahydrocannabinol
(Δ9-THC), are often immunosuppressive, there is an interest in determining the effect
of marijuana compounds on initial anti-HIV immune responses. The objectives
of the studies are to determine if Tat-induced antibody production is affected
by cannabinoid compounds. A dendritic cell line (DC2.4) was engineered to express
Tat (DC2.4-Tat) as a surrogate for HIV-infected antigen presenting cell. The
DC2.4-Tat bulk culture was cloned in limiting dilution and several clones expressed
high levels of Tat and the puromycin resistance gene. Expression of Tat in
DC2.4 cells increased cell size and levels of the activation markers, MHC class I,
MHC class II, CD80 and CD86. DC2.4 or DC2.4-Tat was used to stimulate IgM
antibody from splenic B cells. We demonstrated that DC2.4-Tat cells specifically
induced IgM at 7 days and that pre-treating DC2.4-Tat with LPS boosted that response.
Moreover, Δ9-THC pre-treatment of SPLC reduced IgM production at 7
days. Overall, these results suggest that co-incident marijuana use at the time of initial
HIV infection might suppress early anti-HIV immune responses. (Supported in
part by NIH RO1DA07908, R25NS065777, and the 2010 Michigan State
University Summer Research Opportunity Program).
679 GLOBAL AND TEMPORAL GENE EXPRESSION
ANALYSIS OF PERITONEAL MACROPHAGES IN A
MOUSE ALCOHOL BINGE DRINKING MODEL WITH
REDUCED RESISTANCE TO SEPSIS.
X. Deng 1 , B. Cheng 2 , R. Fan 2 and S. Pruett 1 . 1 Department of Basic Sciences,
Mississippi State University, Mississippi State, MS and 2 Cellular Biology and Anatomy,
Louisiana State University Health Sciences Center, Shreveport, LA.
Acute ethanol consumption has been found to be a significant risk factor for mortality
in patients with sepsis. The binge drinking model in mice developed by our
laboratory has produced similar findings. At earlier time (1-2 hr) in our mouse